Chapter 13 Lecture Notes: DNA Function

Chapter 13 Lecture Notes: DNA FunctionI.Transcription (General info)A. Transcription is the synthesis of RNA using DNA as a template.B. Early evidence suggesting an RNA intermediate between DNA and proteins1. DNA was in the nucleus but proteins were made in the cytoplasm2. RNA synthesis in the nucleus was exported to the cytoplasm(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)3. T2 infection of E. coli results in phage specific RNA being producedC. Properties of RNA – Similar to DNA except1. Contains ribose instead of deoxyribose2. Contains uracil instead of thymine3. Single stranded instead of double stranded (although there are regions ofpairing)D. Misc other info1. Each RNA species is complementary to one strand (template strand) of theDNA double helix.(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)2. Upstream vs. downstream: RNA strand has a 5’and 3’end. Upstream refersto “towards the 5’end” and downstream refers to “towards the 3’end”.3. The region of DNA that contains sequences that are the signals fortranscribing a gene are termed promoters.4. 1 refers to the basepair where transcription starts; -x refers to x basepairs 5’to the start site

II.Factors required for transcriptionA. Prokaryotic1. RNA polymerase (enzyme that catalyzes the synthesis of RNA from a DNAtemplate).a) Core enzyme 3 different types of subunits (2α; 1β; 1β’)(1) β - binds incoming nucleotides(2) β’– binds DNA(3) α - helps with enzyme assembly; interacts with othertranscriptional activator proteins; recent work demonstrated that αalso interacts with some DNA sequencesb) Holoenzyme core σ factor (recognizes the promoter)c) σ factors – Initially, people thought that there was only one σ factorthat functioned to direct RNAP to the promoters of genes. Later, differentclasses of σ factors were found. Each σ factor directs RNAP to a differenttype of promoter (differentiated by a specific DNA sequence in thepromoter).(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)2. Accessory transcription activator proteinsa) Can bind to specific DNA sequences and help RNA polymeraseinitiate transcription via protein-protein interactions or by altering thestructure of the DNA.b) Transcription of some promoters requires an accessory transcriptionalactivator; at other promoters, the activators just increase the rate oftranscription but are not absolutely required.3. Template DNA containing gene or genes to be transcribed4. Promoter - The regulatory element that determine when a gene “turned on”(transcribed) or “turned off”. The promoter DNA is located upstream of the geneand contains a sequence which σ factor of RNAP and other transcription factorsbind. Different classes of promoters have different DNA sequences. Deviationsfrom the consensus sequence decrease the level of transcription.PromoterFunction-35 sequenceSigma 70-dependentSigma 32-dependentSigma 28-dependentSigma S-dependentSigma 54-dependentHousekeepingHeat shock stress responseFlagella synthesisStationary phase survivalNitrogen utilization; pilinTTGACATCTCNCCCTTGAACTAAA?CTGGNA (-24)17 bpspacer-10 sequenceTATAATCCCCATNTACCGATAT?TTGCA (-12)

5. Weak promoters (ones that have poor sigma recognition sequences) haveadditional sequences to which transcriptional activators can bind.6. NTPs, Mg2 B. Eukaryotic1. RNA polymerases – Much more complex that prokaryotic RNAP (numerousadditional factors required, multiple polymerases )a) RNAP I – synthesizes ribosomal RNAb) RNAP II – synthesizes messenger RNAc) RNAP III – synthesizes transfer RNA and 1 type of rRNA2. Eukaryotic RNAPs have subunits that are homologous to α, β, and β’ofprokaryotic RNAP; however, eukaryotic RNAP also contain many additionalsubunits.(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)3. Template DNA containing the gene to be transcribed4. Eukaryotic promoters – contain some combination of the followinga) contain a TATA rich region located –25 to -30 from the start oftranscriptionb) Upstream from the TATA region is a variably located sequencecontaining the sequence CCAAT (frequently at –75)c) GC boxd) Some promoters have other sequences located either upstream ordownstream that maximize the level of transcription called enhancers5. NTPs, Mg2

III.Prokaryotic transcriptionA. Initiation1. RNAP scans the DNA looking for promoters.2. σ factor of RNAP binds the corresponding σ factor recognition sequence inthe promoter.3. Recent evidence suggests that at some promoters, the α subunit may bind toAT rich regions upstream of the sigma binding sites.4. RNAP is bound covering approx. 60 basepairs. The DNA is still is a doublehelix (closed complex).5. RNAP unwinds the DNA resulting in open complex formation.6. First nucleotides are added to start RNA chain. Transcriptional initiation hasoccurred!7. Accessory transcription factors may aid in all of the above listed steps.(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)B. Elongation1. Elongation is 5’à 3’2. σ factor is ejected from RNAP after first 2-10 nucleotides are added.3. Much less is known about this step for transcription than initiation. It wasonce believed that elongation occurred at a constant rate; however, recent worksuggests that RNAP may pause during elongation. In fact, pausing is important intermination (see below).

(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)C. Termination (2 types)1. Rho independent: A specific sequence at the end of the gene signalstermination. The sequence is transcribed into RNA and it is the RNA sequencethat is important. This sequence contains numerous Gs and Cs, which forms ahairpin structure, followed by a string of Us.(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)The hairpin destabilizes the DNA:RNA hybrid leading to dissociation of the RNAfrom the DNA.

2. Rho dependent: Rho protein binds to a sequence in the RNA (rut site – notwell characterized). Rho moves along the RNA in the 3’direction until ineventually unwinds the DNA:RNA hybrid in the active site, thereby pulling theRNA away from the DNA and RNAP. Rut sites are located 5’to sites in theDNA that cause RNAP to pause. It is thought that this allows Rho to catch up toRNAP and the RNA-DNA hybrid.(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)IV.Eukaryotic transcriptionA. Initiation and elongation are similar to in prokaryotes; however, there are severalimportant differences.(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki, Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)

B. Termination of transcription in eukaryotes is poorly understood.C. RNA processing1. 5’capping: Occurs early in transcription. Guanosyltransferase adds 5’methyguanosine (Cap) to 5’end of mRNA. The Cap is important for translationinitiation and for export from the nucleus.(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)2. 3’poly(A) tail: AAUAAA sequence in the RNA signals a cleavage event inthe RNA. Poly(A) polymerase then adds 150-200 A residues are added to the 3’end of the mRNA. The poly(A) tail increases the stability of the mRNA ineukaryotes.(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)

As a side note, recent evidence has demonstrated that there are poly(A)polymerases in prokaryotes and that some mRNAs have poly(A) tails.Interestingly though, the polyA tail destabilizes the mRNA in prokaryotes.Some α2-thalassemias (anemia due to imbalance of α and β hemoglobinsubunits) have been attributed to a defect in polyadenylation. Specifically, thereis a mutation in the cleavage site from AAUAAA à AAUAAG.3. Splicing: The primary transcripts often contain intervening sequences(introns) that are removed from the RNA prior to translation by a cleavagereaction catalyzed by snRNPs (small nuclear ribonuclear proteins which containRNA and protein). Frequently, the splicing site in the intron has a GU at the 5’end and an AG at the 3’end. The snRNP aligns these ends in a lariat formation toallow precise splicing.(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)Complexes containing the snRNP, mRNA, and associated proteins are calledspliceosomes.Splicing is important (1) splicing allows variations of a gene and therefore geneproduct to be made (2) it has been suggested that exons correspond to functionalmotifs in proteins and thus the presence of genes that require slicing allows forevolutionary tinkering (3) many viruses have spliced mRNAs and sounderstanding the process may lead to new therapeutic approaches.As an interesting aside, people with systemic lupus erythematosus have antibodiesdirected against snRNP protein subunits. The significance of this is unknown atthis time.

Example of processing of eukaryotic mRNA:(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)D. RNA export: RNA synthesis and processing occurs in the nucleus. The maturemRNA is then transported through the nuclear pores in the nuclear envelope to thecytoplasm. There is a nuclear complex that is involved in the transport. This complexrecognizes the 5’CAP of the mRNA.

V.Translation – General infoA. Translation is the production of a polypeptide (protein) using RNA as a template andtRNA molecules as “adapters” that convert the nucleic acid code to protein code.B. The nucleotides (letters) of RNA formed codons (words) that specify a particularamino acid.C. The tRNA contains an anticodon that is complementary to the codon and carries aspecific amino acid.D. Important elements of the genetic code:1. The code is a triplet code: Each mRNA codon (word) that specifies aparticular amino acid in a polypeptide chain consists of three nucleotides (letters).For example, AAG lysine2. The code is non-overlapping: The mRNA encoding one protein is read insuccessive groups of three nucleotides.3. The code is degenerate: More than one mRNA codon (word) occurs for someamino acids (ie. AAG and AAA are read as both read as lysine)a) Wobble – certain different codons are recognized by the same tRNAsbecause the 3rd base in the codon and the 1st base of the anticodon pair viaa “loose pairing”. This “loose pairing is according to a set of rules knownas the wobble rules.(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,Leontin, Gelbart 1996 by W. H. Freeman and Company. Used with permission.)b) There is more than one tRNA type (therefore more than oneanticodon) for some amino acids.